1. Field of the Invention
The present invention relates to mutant transglutaminase (TG) proteins of actinomycetous origin. Transglutaminase (also simply referred to as “TG”) is widely utilized for food processing and the like since it catalyzes the formation of a cross-linking bond between proteins, resulting in a gel-like substance. TGs which are mutated to improve transglutaminase activity or thermal stability can help to reduce the amount of TG required and can be used at high temperatures, thus making it possible to apply this enzyme to new fields.
2. Brief Description of the Related Art
Transglutaminase is an enzyme that catalyzes the acyl transfer reaction of the γ-carboxamide group in a peptide chain of a protein. When this enzyme is allowed to act on a protein, a reaction to form the ε-(γ-Glu)-Lys cross-linking and a reaction to replace Gln with Glu by deamidation can occur. Transglutaminases of animal origin and those of microbial origin have been reported to date. Those of animal origin are Ca2+-dependent, and distributed in animal organs, skin, blood, and the like. For example, guinea pig liver transglutaminase (K. Ikura et al. Biochemistry, 27, 2898, 1988), human epidermal keratinocyte transglutaminase (M. A. Phillips et al. Proc. Natl. Acad. Sci. U.S.A., 87, 9333, 1990), human blood coagulation factor XIII (A. Ichinose et al, Biochemistry, 25, 6900, 1986), and the like have been reported. TGs of microbial origin that are non-Ca2+-dependent have been reported in bacteria of the genus Streptoverticillium. Examples include Streptoverticillium griseocameum IFO 12776, Streptoverticillium cinnamoneum sub sp. cinnamoneum IFO 12852, Streptoverticillium mobaraense IFO 13819, and the like. The transglutaminase found in a culture supernatant of a variant of Streptoverticillium mobaraense, in particular, is referred to as MTG (Microbial Transglutaminase). Furthermore, a Ca2+-independent transglutaminase has also been discovered in Streptomyces lydicus NRRL B-3446 (JP-A-H10-504721). It has been found, as a result of peptide mapping and gene structural analysis, that the primary structures of the transglutaminases produced by these microorganisms are not homologous at all with those of animal origin (EP-A-0481504 A1).
MTG is a monomeric protein consisting of 331 amino acids, and having a molecular weight of about 38,000 (T Kanaji et al, Journal of Biological Chemistry. 268, 11565, 1993). Because MTG is often purified from a culture of one of the aforementioned microorganisms and the like, there have been problems with respect to the amount supplied, efficiency, and the like. Attempts have also been made to produce transglutaminase by genetic engineering techniques. A method based on secretory expression by Escherichia coli (E. coli), yeast and the like (JP-A-H5-199883), a method wherein Escherichia coli is allowed to express MTG as a protein inclusion body after which this inclusion body is solubilized with a protein denaturant, treated to remove the denaturant, and then reconstituted to produce active MTG (JP-A-H6-30771), and a method for secretory expression of MTG using Corynebacterium glutamicum (WO2002/081694) have been reported. Unlike transglutaminases of animal origin, MTG and other transglutaminases of microbial origin are Ca2+-independent, and are hence utilized for production of gelled foods such as jellies, yogurt, cheese, or gel-based cosmetics and the like, as well as for improving the quality of meat and the like (JP-A-64-27471). MTG is also utilized for production of raw materials for heat-stable microcapsules, carriers for immobilized enzymes and the like, and so is industrially highly useful. Regarding enzymatic reaction conditions, a gelled food, for example, does not set if the enzymatic reaction time is short, and conversely, if the reaction time is too long, the gelled food becomes too hard to be a commercial product. Hence, when MTG is utilised for production of gelled foods such as jellies, yogurt, cheese, or gel-based cosmetics and the like, or to improve the quality of meat and the like, the desired product is prepared by adjusting substrate and enzyme concentrations, reaction temperature, and reaction time. However, as foods, reagents and the like, which can be produced by utilizing MTG, have become increasingly diverse, in some instances the desired product cannot be prepared solely by adjusting concentrations, temperature, time and the like; therefore, there is a need to modify the enzymatic activity of MTG.
Wild-type MTG (wild-type MTG means an MTG that occurs naturally and has not undergone a modification in its amino acid sequence) is known to be stable at pH between about 4 and 10, and can be stable over a relatively broad range of pH, but under extremely acidic or alkaline conditions, wild-type MTG loses its activity. The optimum temperature for wild-type MTG is about 50° C., and it also loses activity if the temperature is too high. Even at temperatures lower than the optimum temperature, incubation for a long time can result in reduced enzymatic activity. Therefore, a transglutaminase which has been mutated to improve pH stability, thermal stability, and the like will be expected to allow for the use of transglutaminase for new purposes.
MTG has been utilised mainly in the food area so far. Feasibility of application in a wide variety of uses, including textiles, chemical products (photographic films, tanning), feeds, cosmetics, and pharmaceuticals, has been suggested.
In the textile area, wool modification with transglutaminase has been reported. Specifically, it is known that by treating wool with transglutaminase, anti-shrinkage quality, anti-pilling quality, and hydrophobicity can be conferred while maintaining the original texture (JP-A-H3-213574). When transglutaminase is used in wool manufacture and processing, a reaction to keratin at a high temperature in a short time would increase throughput per unit time and improve production efficiency, and therefore, is thought to be industrially useful.
Tanning refers to a processing wherein an animal hide/skin is subjected to a series of treatments and processing consisting of a plurality of steps to render the hide/skin a durable, flexible leather; this processing is achieved by cross-linking the collagen of the hide/skin with hexavalent chromium. Because hexavalent chromium is harmful and the release thereof into the environment is unwanted, there is strong demand for the development of an alternative method. Regarding the utilisation of transglutaminase for tanning, U.S. Pat. No. 6,849,095 discloses that a transglutaminase of microbial origin can be used for tanning, but discloses no examples of actually allowing the transglutaminase to act on a hide/skin; a transglutaminase has not yet been in practical application for this purpose. Because cross-linking with hexavalent chromium takes place at pH 3 to 4, transglutaminase is also likely able to react at this pH, but because MTG is labile to acidity, actual application is difficult.
Hence, in the case of applications such as textile processing and tanning, it is desirable that the thermal stability (i.e., heat resistance) of transglutaminase be improved to complete the reaction at high temperature in a short time, and that the pH stability be improved to allow the reaction in to proceed under acidic conditions.
As stated above, improving the thermal and pH stability of transglutaminase is possible by modification, and thereby improving the enzymatic activity of the transglutaminase. For example, improving the thermal stability may allow for a possible increase in reaction rates and the like. Also, improving the pH stability will allow the enzymatic reaction to proceed over a broader range of pH values, and the product can be stored also over a broader range of pH values. This will also be advantageous in industrialization.
To modify the heat resistance and/or pH stability of transglutaminase, it is necessary to prepare a mutant of the transglutaminase and evaluate the activity and the like thereof. To prepare a mutant, it is necessary to manipulate the wild-type gene; therefore, a recombinant protein must be prepared. In the case of MTG, a secretory expression system using Corynebacterium glutamicum has been established (WO2002/081694).
To increase the stability of a protein, it is generally possible to introduce a non-covalent bond such as a hydrogen bond, a covalent bond such as a disulfide bond, or an electrostatic or hydrophobic interaction to enhance the packing of the hydrophobic core within the molecule. Other methods include stabilizing the a helix in the secondary structure, or removing factors that make the structure of the protein unstable. To increase the stability of a protein by introducing a disulfide bond, it is necessary to determine positions suitable for introducing cysteine. In the case of MTG, mutated transglutaminases whose heat resistance and/or pH stability is improved by introducing a disulfide bond have been created (WO2008/099898).
WO2008/099898 discloses mutated transglutaminases prepared by introducing a disulfide bond into the wild-type transglutaminase, and the like. For example, WO2008/099898 discloses proteins possessing transglutaminase activity which have at least a set of mutations selected from among:                a) substitution of 7-position and 58-position to cysteine,        b) substitution of 46-position and 318-position to cysteine,        c) substitution of 93-position and 112-position to cysteine,        d) substitution of 106-position and 213-position to cysteine,        e) substitution of 160-position and 228-position to cysteine,        f) substitution of 2-position and 282-position to cysteine,        g) substitution of 2-position and 283-position to cysteine,        h) substitution of 3-position and 283-position to cysteine, and        i) substitution of 17-position and 330-position to cysteine,in the sequence of MTG, polynucleotides that encode the proteins, recombination vectors including one of the polynucleotides, host cells transformed with one of the vectors, a method of producing a transglutaminase by culturing the host cells, and the like. This reference also discloses a method for processing a substrate protein using the described mutated transglutaminase.        
However, although some improvements in the heat resistance and/or pH stability were observed in mutated transglutaminases containing disulfide bonds, further improvement of the heat resistance is desired so that the proteins can endure high temperatures used for textile processing and the like.
To modify the enzyme activity of a mutated transglutaminase containing one or more disulfide bonds, it is necessary to prepare a mutant thereof and evaluate the activity and the like thereof. To prepare a mutant, it is necessary to manipulate the wild-type gene; therefore, a recombinant protein must be prepared. In the case of MTG, a secretory expression system using Corynebacterium glutamicum has been established (WO2002/081694).
Although C. K. Marx et al, Journal of Biotechnology, 136 (3), p. 156-162, September 2008 describes some MTG mutants, the heat resistances of these mutants are inadequate for use at high temperatures like those used for textile processing and the like.